1. Field of the Invention
The field of the present technology relates to chemotherapeutics. Particularly, the field relates to the topical or injected treatment of cancer cells with specific pharmacological materials.
2. Background of the Art
Cervical cancer is the second most common cancer diagnosis in women and is linked to high-risk human papillomavirus infection 99.7% of the time. Currently, 12,000 new cases of invasive cervical cancer are diagnosed in US women annually, resulting in 5,000 deaths each year. Furthermore, there are approximately 400,000 cases of cervical cancer and close to 200,000 deaths annually worldwide. Human papillomaviruses (HPVs) are one of the most common causes of sexually transmitted disease in the world. Overall, 50-75% of sexually active men and women acquire genital HPV infections at some point in their lives. An estimated 5.5 million people become infected with HPV each year in the US alone, and at least 20 million are currently infected. The more than 100 different isolates of HPV have been broadly subdivided into high-risk and low-risk subtypes based on their association with cervical carcinomas or with benign cervical lesions or dysplasias.
A number of lines of evidence point to HPV infections as the etiological agents of cervical cancers. Multiple studies in the 1980's reported the presence of HPV variants in cervical dysplasias, cancer, and in cell lines derived from cervical cancer. Further research demonstrated that the E6-E7 region of the genome from oncogenic HPV 18 is selectively retained in cervical cancer cells, suggesting that HPV infection could be causative and that continued expression of the E6-E7 region is required for maintenance of the immortalized or cancerous state. The following year, it was demonstrated that the E6-E7 genes from HPV 16 were sufficient to immortalize human keratinocytes in culture. It was also demonstrated that although E6-E7 genes from high risk HPVs could transform cell lines, the E6-E7 regions from low risk, or non-oncogenic variants such as HPV 6 and HPV 11 were unable to transform human keratinocytes. More recently, HPV 16 and 18 infection by in situ hybridization and E6 protein expression by immunocytochemistry in 623 cervical tissue samples were examined at various stages of tumor progression and found a significant correlation between histological abnormality and HPV infection.
Human papillomaviruses characterized to date are associated with lesions confined to the epithelial layers of skin, or oral, pharyngeal, respiratory, and, most importantly, anogenital mucosae. Specific human papillomavirus types, including HPV 6 and 11, frequently cause benign mucosal lesions, whereas other types such as HPV 16, 18, and a host of other strains, are predominantly found in high-grade lesions and cancer. Individual types of human papillomaviruses (HPV) which infect mucosal surfaces have been implicated as the causative agents for carcinomas of the cervix, anus, penis, larynx and the buccal cavity, occasional periungal carcinomas, as well as benign anogenital warts. The identification of particular HPV types is used for identifying patients with premalignant lesions who are at risk of progression to malignancy. Although visible anogenital lesions are present in some persons infected with human papillomavirus, the majority of individuals with HPV genital tract infection do not have clinically apparent disease, but analysis of cytomorphological traits present in cervical smears can be used to detect HPV infection. Papanicolaou tests are a valuable screening tool, but they miss a large proportion of HPV-infected persons due to the unfortunate false positive and false negative test results. In addition, they are not amenable to worldwide testing because interpretation of results requires trained pathologists. Because of the limited use and success rate of the Papanicolaou test, many HPV-infected individuals fail to receive timely diagnosis, a problem that precludes efforts to administer treatment prior to the appearance of clinical symptoms. A significant unmet need exists for early and accurate diagnosis of oncogenic HPV infection as well as for treatments directed at the causative HPV infection, preventing the development of cervical cancer by intervening earlier in disease progression.
Because treatments are usually administered after the onset of clinical symptoms, current treatment paradigms are focused on the actual cervical dysplasia rather than the underlying infection with HPV. Women are screened by physicians annually for cervical dysplasia and are treated with superficial ablative techniques, including cryosurgery, laser ablation and excision. As the disease progresses, treatment options become more aggressive, including partial or radical hysterectomy, radiation or chemotherapy. All of these treatments are invasive and carry the possibility or guarantee of permanent infertility. In addition, surgical removal of tissue may not guarantee that all infected cells have been eliminated due to the fact that some transformed cells may not yet be displaying the morphological changes associated with HPV infection.
More recently, research has focused on nonsurgical alternatives for the treatment of HPV infection and cervical cancer. Various DNA and protein treatments designed to induce apoptosis in cells may reduce the number of cancerous cells, but may also induce apoptosis in healthy cells. Topoisomerase inhibitors such as irinotecan (Camptosar®) and inhibitors of thymine production such as fluorouracil (Fluoroplex®, Efudex®, Adrucil®) nonspecifically prevent cell division. While these treatments are beneficial therapies for the treatment of a variety of cancers, they pose significant risk to healthy cells and fail to specifically target HPV infected cells.
Published US Patent Application 20040259876 (Shiraishi et al.) describes methods of synthesis of medicinal materials that may be useful in certain oncologic environments using phenyl boric acid during the synthesis.
DU-145 (human, prostrate, carcinoma); DSMZ ACC 261
Morphology: epithelial-like adherent cells growing as monolayers human prostate carcinoma established from the tumor tissue removed from the metastatic central nervous system lesion of a 69-year-old man with prostate carcinoma in 1975 confirmed as human with IEF of AST, MDH, NPViruses: ELISA: reverse transcriptase negative; PCR: EBV−, HBV−, HCV−, HHV-8−, HIV−, HTLV-I/II−Depositor: obtained from DKFZ Tumorbank, Heidelberg, GermanyGeneral RestrictionsProperties: cytokeratin+, cytokeratin-7+, cytokeratin-8+, desmin−, endothel−, GFAP−, HMB-45−, neurofilament−, vimentin+Available in the following: German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig). 90% RPMI 1640+10% FBS split confluent culture 1:3 to 1:5 every 2-3 days using trypsin/EDTA; seed out at ca. 2-3×106 cells/80 cm 2 at 37 C with 5% CO2 cell harvest of about 35×106 cells/175 cm 2; doubling time of about 30-40 hours frozen with 70% medium, 20% FBS, 10% DMSO at about 2-3×106 cells/ampoule; negative in DAPI, microbiological culture, RNA hybridization, PCR assays. Fingerprint: unique DNA profile using multiplex PCR at D1S80, D2S44, D17S5 and ApoB Cytogenetics: human hypotriploid karyotype with 12% polyploidy; 62(58-65)<3n>X, −X/Y, −X/Y, −2, −3, +5, −8, −10, −13, +15, +15, −16, −18, −19, −20, −21, −22, +3mar, del(1)(p31), del(2)(p11), i(5p), del(6)(q22), del(9)(p12), del(11)(q23), der(12)t(11;12)(q11;p11), add(13)(q33), add(13)(q33), add(15)(p11)x2, add(16)(q24); closely resembles reported karyotype. Availability in cell line catalogues: ATCC HTB 81.
“The Surface of Prostate Carcinoma DU145 Cells Mediates the Inhibition of Urokinase-type Plasminogen Activator by Maspin,” Richard McGowen, Hector Biliran, Jr., Ruth Sager2 and Shijie Sheng3 (Department of Pathology, Wayne State University School of Medicine, Detroit, Mich. 48201 [R. M., H. B., S. S.], and Division of Cancer Genetics, Dana-Farber Cancer Institute, Boston, Mass. 02115 [R. S.]) describes that Maspin is a novel serine protease inhibitor (serpin) with tumor suppressive potential in breast and prostate cancer, acting at the level of tumor invasion and metastasis. It was subsequently demonstrated that maspin inhibits tumor invasion, at least in part, by inhibiting cell motility. Interestingly, in cell-free solutions, maspin does not inhibit several serine proteases including tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA). Despite the recent biochemical evidence that maspin specifically inhibits tissue-type plasminogen activator that is associated with fibrinogen or poly-L-lysine, the molecular mechanism underlying the tumor-suppressive effect of maspin remains elusive. The goal of this study was to investigate the effect of maspin on cell surface-associated uPA. In our experimental system, we chose prostate carcinoma DU145 cells because these cells mediate plasminogen activation primarily by uPA, as shown by two different colorimetric enzyme activity assays. Purified recombinant maspin produced in baculovirus-infected Spodoptera frugiperda Sf9 insect cells [rMaspin(i)] binds specifically to the surface of DU145 cells, inhibits the DU145 cell surface-bound uPA, and forms a stable complex with the uPA in DU145 cell lysate. The inhibitory effect of rMaspin(i) on cell surface-bound uPA was similar to that of an uPA-neutralizing antibody and was reversed by a polyclonal antibody against the reactive site loop sequence of maspin. The Ki value for rMaspin(i) in cell surface-mediated plasminogen activation was 20 nM, which was comparable to the Ki values for plasminogen activator inhibitor 1 and plasminogen activator inhibitor 2, respectively. Furthermore, the proteolytic inhibitory effect of rMaspin(i) was quantitatively consistent with its inhibitory effect on the motility of DU145 cells in vitro. Our data demonstrate an important role for the prostate carcinoma cell surface in mediating the inhibitory interaction between rMaspin(i) and uPA. Thus, future maspin-based therapeutic strategies may prove useful in blocking the invasion and metastasis of uPA-positive prostate carcinoma.
TKG 0604::DU145; ID:
TKG 0604; Cell name: DU145; Animal: Human; Scientific name: Homosapiens; Tissue: Prostate carcinoma brain metastasis. Passage method: 0.02% EDTA-PBS; Life Span: Infinite; Morphology: Epithelial-like; Medium: RPMI-1640 plus 10% FBS or Eagle's MEM plus 10% FBS; Characteristics: This cell line was established from a lesion in the brain of a patient (69 ear-old, Caucasian, blood type O) with widespread brain metastasis of prostate carcinoma and a 3-year history of lymphocyteic leukemia. Tumorigenic in nude mouse. Established by: K. R. Stone; References: Int. J. Cancer, 21, 274-281, 1978. Cancer Res., 37, 4049-4058, 1977.
Anisomycin (Anisomycin; Br. J. Cancer, 2003 Nov. 4; 87 (10):1188-94) activates JNK and sensitises DU 145 prostate carcinoma cells to Fas mediated apoptosis. Curtin J F, Cotter T G. Department of Biochemistry, University College Cork, Lee Maltings, Prospect Row, Cork, Ireland. Treatment of the hormone refractory prostate cancer cell line DU 145 with sublethal concentrations of chemotherapeutic drugs has been reported to sensitise these cells to Fas mediated apoptosis. However, the mechanism by which this occurs has not been determined. Our group has shown that inhibition of JNK activity completely abrogates the effects of chemotherapeutic drugs. Using anisomycin, a potent JNK agonist, we have demonstrated a role for JNK in Fas mediated apoptosis in DU 145 cells. Inhibition of Caspase 8 and Caspase 9 completely inhibits this process which suggests that DU 145 cells require mitochondrial amplification of the Fas apoptotic signal. Furthermore, we have shown that inhibition of Fas mediated apoptosis is an early event in DU 145 cells, occurring upstream of Caspase 8 cleavage. It is hoped that identifying the target of JNK will allow novel therapies to be developed for the treatment of hormone refractory prostate cancer. Such therapies are especially important because no single or combined treatment to date has significantly prolonged survival in patients with hormone refractory prostate cancer. Copyright 2002 Cancer Research UK
Characterization of Prostate Cancer DU145 Cells Expressing the Recombinant Androgen Receptor; Authors: Scaccianoce E.; Festuccia C.; Dondi D.; Guerini V.; Bologna M; Motta M.; Poletti A. Source: Oncology Research Incorporating Anti-Cancer Drug Design, Volume 14, Number 2, 2003, pp. 101-112(12) Publisher: Cognizant Communication Corporation:
Prostate cancer (PC) develops as a consequence of abnormal androgenic stimulation. Unfortunately, most of the PC cell lines are androgen independent (like DU145), or express mutated forms of androgen receptor (AR). We have produced and characterized a new stably transfected PC line expressing the AR (DU145-AR). Untreated DU145-AR cells showed a lower proliferation rate than mock transfected cells, but responded to testosterone treatment. PSA mRNA, undetectable in mock DU145 cells, was present and upregulated by testosterone in DU145-AR. About 5% of DU145-AR cells showed modification of morphology and enriched of f-actin after testosterone treatment. Moreover, in DU145-AR plasminogen activator (PA) activity and secreted urokinase type plasminogen activator (uPA) protein were lower than in AR negative cells; again testosterone induced PA activity and uPA protein only in DU145-AR. These results indicate that, in general, the effects of unactivated AR is to suppress function(s) in DU145 cells and the addition of testosterone restores the normal properties associated with the untransfected cells. Some of the effects described may thus be mediated by a ligand-independent activation of AR in DU145 cells.
Downregulation of c-FLIP Sensitizes DU145 Prostate Cancer Cells to Fas-Mediated Apoptosis; Author(s): Marc L. Hyer, Sunil Sudarshan, Youngsoo Kim, John C. Reed, Jian-yun Dong, David A. Schwartz and James S. Norris; Article Vol: 1|Issue: 4|July/August 2002|pgs: 401-406|Research Paper; Abstract:
Although DU145 prostate cancer cells are resistant to exogenously applied Fas agonist CH-11 (anti-Fas monoclonal antibody), Fas-resistance can be overcome using a FasL expressing adenovirus (AdGFPFasLTET) (Hyer et al., Mol. Therapy, 2: 348-58, 2000). The purpose of this study was to try to understand why DU145 cells are resistant to CH-11 and determine the signaling pathway utilized by AdGFPFasLTET to induce apoptosis in these Fas-resistant cells. Using immunoblot analysis, we show that AdGFPFasLTET is capable of initiating the classic Fas-mediated apoptotic pathway in DU145 cells, which includes activation of caspases-8, -3, -7, and -9, BID cleavage, cytochrome c release from mitochondria, and PARP cleavage. In contrast, CH-11 binds to Fas, but is unable to transmit the death signal beyond the plasma membrane suggesting a block at the DISC (death inducing signaling complex). The anti-apoptotic protein c-FLIP (cellular Flice-like inhibitory protein), which has been shown to inhibit Fas-mediated apoptosis at the DISC, was down-regulated following AdGFPFasLTET treatment prompting us to investigate its role in inhibiting CH-11-induced cell death. Using c-FLIP anti-sense oligonucleotides to down-regulate c-FLIP we sensitized DU145 cells to CH-11-induced apoptosis. These data suggest that c-FLIP may play a critical role in regulating Fas-mediated apoptosis in prostate cancer cells and that modulation of c-FLIP may enhance Fas signaling based therapies.
All of the references cited herein are incorporated herein by reference in their entirety for their technical disclosure of materials, methods, protocols, and related technology. All publications cited herein are incorporated by reference in their entirety and for all purposes.